Summary This proposal is a revised application that examines regulation of human macrophages by the intracellular voltage-gated sodium channel, NaV1.5. Macrophages play a central role in the pathogenesis of intracellular bacterial and viral infections as seen in tuberculosis (TB) and HIV, and they directly mediate tissue injury in autoimmune diseases such as multiple sclerosis (MS). Studies examining unique features of human macrophages at the cellular level are required to develop specific anti-macrophage therapies for human disease. Recently, we identified two novel variants of voltage-gated sodium channels, NaV1.5 and NaV1.6, that are expressed in human macrophages and may represent novel pharmacologic targets. In this revised application, we focus on the role of the NaV1.5 channel in human macrophage function. Macrophage NaV1.5 is unique to human macrophages and is expressed intracellularly on the late endosome. Preliminary data suggest that macrophage NaV1.5 may act as a pathogen biosensor to enhance inflammatory responses and that it regulates macrophage endosomal acidification, phagocytosis, and inflammatory signaling. The hypothesis of this proposal is that the human macrophage splice variant of the voltage- gated sodium channel NaV1.5 has novel molecular properties that regulate intracellular processing and signaling. The revised Specific Aims of this proposal are to analyze: I.) The electrophysiologic properties of macrophage NaV1.5; II.) NaV1.5 regulation of macrophage processing; and III.) NaV1.5 regulation of intracellular signaling. The experimental approach to these aims will be multi-disciplinary and will include electrophysiologic, microscopic, fluorometric, and biochemical techniques. To characterize how the channel is activated, novel methods of single channel recordings in isolated endosomes will be combined with more traditional methods of whole cell patch clamp recordings in transfected cells that over-express NaV1.5. To assess the cellular immune function of the channel, Bacillus Camille-Guerin (BCG) will be used as a model of intracellular mycobacteria infection, and NaV1.5-dependent endosomal processing and intracellular signaling will be assessed by live cell microscopy in addition to standard approaches. These studies represent one of the first electrophysiologic characterizations of endosomal channels and subcellular voltage-gated sodium channels. Since this work will be performed in primary human cells and cell lines, these studies are a unique opportunity to characterize novel properties of human macrophage function and to translate these findings to developing anti-macrophage therapeutics and new diagnostic techniques. Voltage-gated sodium channels are attractive pharmacologic targets because their function can be blocked by low molecular weight, orally active drugs that are currently used clinically to treat epilepsy. Related novel agents that specifically modulate macrophage sodium channels also could be developed. These sodium channel-specific agents may represent a more economical choice for immune modulation because of their relatively low cost as compared with many current monoclonal antibody-based biologic treatments. One goal of these studies is to develop therapeutic strategies to inhibit macrophage NaV1.5 selectively to treat chronic inflammatory conditions such as TB, HIV, and MS in veterans. A second goal is to determine whether or not expression and activity of these macrophage proteins can serve as a biomarker of inflammatory disease activity to aid diagnosis and permit optimization of treatment.